Specific antibody fragments for the human carcinoembryonic antigen (cea)

ABSTRACT

The invention refers to mono- and divalent (diabody) single chain Fv (scFv) antibody fragments, obtained by recombinant DNA techniques from the anti-carcinoembryonic antigen (CEA) monoclonal antibody (Mab) CB/ior-CEA.1. This antibody has high affinity for CEA and is employed in the diagnosis and follow-up of human colorectal tumors. As the original Mab, the monovalent fragment and the diabody exhibit high affinity for human CEA and recognize an epitope dependent of carbohydrate conservation. The monovalent scFv fragment and the diabody have affinity constants for CEA of (5.0±0.4)×10 9  L mol −1  and (2.8±0.3)×10 10  L mol −1 , respectively. These two fragments do not show cross reactivity with human normal cells and tissues, exception made of the normal colonic mucosa, where CEA is occasionally present. The fragments can be produced through the expression in recombinant microorganisms, starting from the cloning of the encoding variable region nucleic acid sequences obtained from the hybridoma that produces Mab CB/ior-CEA.1. As the original Mab, the monovalent scFv and the diabody have the ability to identify in vivo cells that produce human CEA and grow as tumors in mice. The monovalent scFv and the diabody have a molecular size 5 and 2.5 times lower, respectively, than the mouse Mab, and do not have Fc domains, fact this that confers them the potential to better penetrate tissues in vivo and to be less immunogenic in man.

TECHNICAL FIELD

The present invention is related to the Branch of Immunology, and inparticular it refers to antibody fragments of the single chain Fv type,in its mono- and divalent (diabody) forms, obtained by recombinant DNAtechniques starting from a mouse monoclonal antibody of proved clinicalefficacy, that is specific for the human carcinoembryonic antigen.

BACKGROUND OF THE INVENTION

The carcinoembryonic antigen (CEA) is a 180 kDa glycoprotein, secretedpreferably by the cells of gastrointestinal human tumors y othercarcinomas, even though it can also be detected in some non-malignanttissues as the colonic mucosa. Its physiological role has not beentotally elucidated, and up to the moment it is believed it is associatedin some way to the processes of cell adhesion (Gold P, Freedman S O.Journal of Experimental Medicine 122: 467; 1965; Zimmermann W et al.PNAS USA 84: 2960-2964; 1987; Paxton R J et al. PNAS USA 84: 920-924,1987; Beauchemin N et al. Molec. Cellular Biol. 7: 3221-3230, 1987; GoldP, Goldenberg N A. M J M 3:46-66, 1997).

The CEA is a member of the immunoglobulin superfamily, due to itsstructure characterized by repetitive domains (Oikawa S et al. BBRC 144:634-642, 1987; Thompson J, Zimmermann W. Tumor Biology 9: 63-83, 1988;Hammarstrom S. Seminars in Cancer Biology 67-81, 1999). The CEA has ahigh homology with other molecules of this superfamily, such as the NCA,the meconium antigen, the Biliar Glycoprotein type A, and theGlycoprotein b specific of pregnancy (von Kleist S, Burtin P.Immunodiagnosis of Cancer. Marcel Dekker. 322-341, 1979; Buchegger, F.et al. Int. J. Cancer 33; 643-649, 1984; Matsuoka Y et al. Cancer Res.42:2012-2018, 1982; Svenberg T. Int. J. Cancer 17:588-596,1976).

The elevation of circulating CEA levels is considered since many yearsago as one of the best indicators of a possible relapse and/ormetastases, in patients submitted to surgery for primary colorectaltumors that express this antigen (Gold P, Goldenberg N A. MJM 3:46-66,1997). The measurement of circulating CEA has extended also as a methodfor the follow-up of other human carcinomas (breast, lung), in the casesin which significant pre-surgery levels of this tumor marker have beendemonstrated (Gold P, Goldenberg N A. MJM 3:46-66,1997).

Since the discovery of the technology for generation of monoclonalantibodies (Mab; Kohler G, Milstein C. Nature 256:52-53, 1975), theimmunoassays for the measurement of circulating CEA have improved inspecificity and their use has widely extended.

The CEA has been studied also since many years ago as a possible “celltarget” in order to specifically direct radioactive isotopes for in vivodiagnostics (Goldenberg D M Int. J. of Biol. Markers 7; 183-188, 1992)and in situ radiotherapy (Ledermann et al., Int. J. Cancer 47; 659-664,1991). Its use has also been foreseen to target toxins, drugs, and otherbioactive products towards the tumor cells (Bagshawe K D. Drug Dev. Res.34:220-230, 1995).

The anti-CEA antibodies have been the main vehicles used for suchpurposes, starting with polyclonal preparations, to be followed later onby mouse Mab, their Fab fragments, antibody fragments obtained bygenetic engineering from mouse Mab, and more recently, from libraries ofmurine and human antibodies displayed in filamentous phage (HammarstromS et al. Cancer Res. 49, 4852-4858, 1989; Hudson P J Curr. OpinionImmunology 11:548-557, 1999; Griffiths A D et al. EMBO J. 12, 1993;725-734; Griffiths A D et al. EMBO J. 13 3245-3260, 1994; WO93/11236;Chester K et al 1995, WO 95/15341; Allen D J et al. 1996, U.S. Pat. No.5,872,215).

The expression of antibodies and antibody fragments in prokaryotic cellslike E. coli, and in other microorganisms is well established in the art(Pluckthun, A. Bio/Technology 9: 545-551, 1991; Gavilondo J, Larrick JW. Biotechniques 29: 128-132, 134-136, 2000). The expression ofantibodies and antibody fragments in superior eukaryotic cells inculture is also know for those skilled in the art (Reff M E. Curr.Opinion Biotech. 4: 573-576, 1993; Trill J J et al. Curr. OpinionBiotech 6: 553-560, 1995).

The mouse Mab denominated indistinctively as CB-CEA.1 or ior-CEA.1(referred hereafter as CB/ior-CEA.1) is known from the state of the art.This Mab has a high specificity for human CEA, has no undesiredcross-reactions with molecules such as NCA, nor recognizes normaltissues, exception made of the cells of the normal colon epithelium,where CEA can be commonly found polarized (Tormo B et al. APMIS 97:1073-1080, 1989). This Mab has very high affinity for CEA (Pérez L etal. Applied Biochem. Biotechnol. 24: 79-42, 1996). This Mab labeled with^(99m)Tc has been successfully employed in the diagnosis and follow upof human colorectal tumors. The clinical studies of radioimmunodetectionshowed that it has 91.3% sensitivity, 77.1% specificity, and 82.8% ofpositive predictive value (Oliva J P et al. Rev Esp Med Nucl. 13:4-10,1994). This makes it superior in performance with respect to the onlyother anti-CEA monoclonal antibody employed clinically in the World atpresent for such purposes, the CEA-Scan (^(99m)Tc-Arcitumomab) fromImmunomedics (Morris Plains, N.J., USA).

The development of a single chain Fv (scFv) antibody fragment, obtainedthrough the polymerase chain reaction (PCR) from RNA extracted from thehybridoma that produces the Mab CB/ior-CEA.1, was reported in 1992(Ayala M et al. Biotechniques 13: 790-799, 1992). In the experimentalstrategy followed, the amplification of the variable domains ofCB/ior-CEA.1 was done with degenerate oligonucleotides for the frameworkregions of both variable domains. The scFv was produced in E. coli anddemonstrated recognition of CEA in ELISA and in cytochemistry studies,but with a affinity for the immobilized antigen 200 times lower than theFab obtained by the natural way (Pérez L et al. Applied Biochem.Biotechnol. 24: 79-82, 1996). This same fragment scFv was cloned,expressed, and produced in Pichia pastoris (Freyre F M et al. JBiotechnol. 76(2-3):157-163, 2000) without any improvements in affinityfor human CEA, and the studies conducted in experimentation animals withthe radiolabeled fragment indicated an anomalous biodistribution(Pimentel G J et al. Nucl Med Commun. 22:1089-94, 2001), that motivatednot to continue its further development.

ESENCE OF THE INVENTION

The present invention refers to single chain Fv (scFv) antibodyfragments, in its mono- and divalent (diabody) forms, obtained by DNArecombinant techniques staring from the anti-carcinoembryonic antigen(CEA) monoclonal antibody CB/ior-CEA.1 (Tormo B et al. APMIS 97:1073-1080, 1989). This Mab has very high affinity for CEA (Pérez L etal. Applied Biochem. Biotechnol. 24: 79-82, 1996) and has beensuccessfully employed in the diagnosis and follow-up of human colorectaltumors (Oliva J P et al. Rev Esp Med Nucl. 13:4-10, 1994). The scFvmonovalent and diabody fragments reported in the present invention canbe produced through their expression in recombinant microorganisms, asbacteria and yeast. As the original Mab, the scFv monovalent and diabodyfragments are specific for an epitope of human CEA that depends on theconservation of the carbohydrates and exhibit high affinities for thisantigen. The scFv monovalent and diabody fragments have a recognitionpattern in vitro of human normal and tumor cells and tissues similar tothe original Mab and, as this, once radiolabeled, they have the capacityto identify tumor cells that express human CEA growing in athymiccongenital mice. The scFv monovalent and diabody fragments have no Fcdomains and have lower molecular size that the mouse Mab, thisconferring them the potential to better penetrate the tissues in vivoand to be less immunogenic when applied to humans for diagnostic ortherapeutic purposes.

The scFv monovalent and diabody fragments reported in this inventionhave important differences in aminoacids in the heavy chain (VH, andlight chain (VL) variable domains, with respect to other scFv previouslydeveloped from the same Mab, and surpass it in affinity for CEA, inperformance for the recognition of cells and tissues, and in efficacyfor the localization of tumors that produce human CEA growing in vivo inmice.

The recombinant scFv monovalent and diabody fragments reported in thisinvention were developed using PCR, and cloning and expressiontechniques in recombinant microorganisms, starting from the RNAextracted from the CB/ior-CEA.1 hybridoma. Sets of oligonucleotidesdifferent from those used to obtain a previously reported scFv (Ayala etal. Biotechniques 13: 790-799, 1992), were employed for theamplification and isolation of the base sequences encoding the Mab VHand VL domains. In the invention it is shown that the new monovalent anddiabody scFv have important differences in the aminoacid sequences ofthe VH and VL domains, with respect to a scFv previously obtained, andthat these take the form of 16 aminoacids in the frameworks 1 (FR1) and3 (FR3) and in the complementary determinant region 2 (CDR2) of the VHdomain, different with respect to the scFv previously obtained, and 3aminoacids between the FR1 and FR3 of the VL domains, different withrespect to the scFv previously obtained. This indicates that thesedomains have a different clonal origin with respect to those reported inAyala et al. Biotechniques 13: 790-799, 1992. In the case of thediabody, this one also differs from the scFv previously obtained in thesize and aminoacidic composition of the union segment (linker) that isemployed in the fabrication of the scFv-type molecule.

The changes reflect surprisingly in the biochemical and biologicalproperties of the new fragments, and provide them with a behavior verysimilar to the Mab CB/ior-CEA.1, and very much superior to that of thepreviously reported scFv. The new monovalent scFv fragment, that has alinker identical to the previously reported scFv (Ayala et al.Biotechniques 13: 790-799, 1992), but the aforementioned aminoacidchanges in the variable domains, has an affinity constant for human CEAvery much higher that the previously reported scFv. Also, the diabodysurpasses both monovalent scFv forms in its affinity constant for humanCEA. The two new scFv monovalent and diabody fragments conserve theproperties of specificity of the original Mab with respect to CEArecognition, identification of tumor cells and tissues, absence of crossreactivity with NCA, and capacity to accumulate selectively in a tumorthat produces human CEA transplanted in mice, all with a very muchsuperior performance than that of the previously obtained scFv.

The two new monovalent and diabody scFv have molecular sizes at least 5and 2.5 times lower than the original Mab, respectively, a fact thatconfers these with the potential to better penetrate tissues and to beless immunogenic in the human being, all of which makes them moreattractive and presumably superior that the original CB/ior-CEA.1 Mab todirect radioisotopes, drugs, toxins, and other bioactive elements totumors that express human CEA.

In the present invention it is shown how it is possible to amplify byPCR the VH and VL domains of the Mab CB/ior-CEA.1 using syntheticoligonucleotides that hybridize in the base sequences that encode forthe signal peptides and constant domains CH1 and Ck. It is also shownthe possibility of assembly of the amplified VH and VL domains, in thisorder, using PCR, and obtaining different forms of scFv fragmentsmanipulating the size of the linker that connects the domains. Using 14aminoacids a monovalent scFv form is originated, and reducing thisnumber to five, a diabody scFv type form is produced.

It is demonstrated in the invention that it is possible to express themonovalent and divalent scFv fragments in the bacteria E. coli and inthe yeast Pichia pastoris, and that these fragments identify in vitrothe human CEA, linked or not to tumor cells, in a specific manner. Inthe present invention it is also demonstrated that the radiolabeledmonovalent and diabody scFv identify in vivo tumor cells that expresshuman CEA and that grow as tumors in mice, exhibiting a behavior verysimilar to that of the Mab CB/ior-CEA.1, and a performance very superiorto the previously obtained scFv. In the present invention methods topurify and characterize the new scFv monovalent and diabody fragments,are also shown.

The antibody fragments described in this invention are useful to beapplied in the diagnosis and therapy of cancer, with the advantages thatthese derive from a Mab of proved clinical efficiency, and that theirlower size and absence of Fc domain allow both a better tissuepenetration, and their use in repeated treatments due to the lessercapacity of induction of a human anti-mouse immunoglobulin response(HAMA; Schroff et al. Cancer Res 45: 879-885, 1985; DeJager et al. Proc.Am. Assoc. Cancer Res. 29:377, 1988). The HAMA responses areinconvenient for the treatment because of the neutralization of thebiological effect of the administered antibody, the consequent doselowering, and because these can cause allergic responses, “serum”sickness, and kidney affections.

Terminology

Antibodies and their Specific Fragments

The terms describe an immunoglobulin of parts thereof with antigenicspecificity, being these natural or produced partially or fully in asynthetic way. The terms also cover any polypeptide or protein that hasa binding domain that would be the binding site of the antibody, orhomologous to it. These can be produced naturally or in a synthetic way,either partially or fully. Examples of antibodies are the differentclasses and subclasses of immunoglobulins, and fragments of these thatcontain one or more antigen binding sites, such as Fab, scFv, Fv and thediabodies.

The antibodies and antibody fragments include any polypeptide thatcomprises an immunoglobulin binding domain, being this natural orproduced synthetically, both fully or partially, and chimeric moleculesthat comprise an immunoglobulin binding domain, or its equivalent, fusedto other polypeptide.

It has been shown that the fragments of a complete antibody can carryout the function of binding antigens. Examples or these bindingfragments are: (i) the Fab fragment that includes the VL, VH, CL and CH1domains of an immunoglobulin; (ii) the Fd fragment, that consists of theVH and CH1 domains; (iii) the Fv fragment, that consist in the VL and VHdomains of a given antibody; (iv) the scFv fragment, where the VH and VLdomains of a given antibody are united with a peptidic linker thatallows the two domains to associate to form an antigen binding site(Bird et al, Science 242: 423-426, 1988; Huston et al, PNAS USA 85:5879-5883, 1988); (v) “diabodies”, multivalent or multispecificfragments constructed in a similar way to scFv but where the small sizeof the linker does not allow the VH and VL domains of the same scFvmolecule to associate among them, and the antigen binding sites formthrough the association of two or more scFv (WO94/13804; Holliger P etal. PNAS USA 90 6444-6448, 1993); (vi) other fragments as the dAb (WardS E et al., Nature 341: 544-546, 1989), isolated CDR regions, F(ab′)₂fragments and bispecific scFv dimers (PCT/U.S.92/09965; Holliger P,Winter G. Current Opinion Biotechnol. 4: 446-449, 1993; de Haard, H etal. Adv. Drug Delivery Rev. 31:5-31, 1998).

The diabodies and scFv can be constructed without Fc regions, using onlythe variable domains, potentially reducing the effects of anti-isotypereactions when administered to humans. They are also particularly usefuldue to their production in E. coli and recombinant yeast. Their sizeinferior to that of a full immunoglobulin provides them with increasedtissues penetration potential.

Antigen Binding Site

This term described the part of an antibody that comprises the area thatspecifically interacts with all the antigen, or part of it. When theantigen is large, an antibody can only bind to a particular part of theantigen, denominated epitope. An antibody-binding site can be given byone or more antibody variable domains. Preferably, an antigen-bindingsite comprises the variable region (or domain) of the light chain (VL)and the variable region (or domain) of the heavy chain (VH) of anantibody.

Specific

Refers to the situation in which an antibody or its fragment does notpresent a significant binding to other molecules different from itsspecific binding pair. This term is also applicable to the case where anantigen binding site is specific for a particular epitope that appearsin a number of related or un-related antigens, in which case the bindingsite would be capable of binding to several antigen that bear theepitope.

DETAILED DESCRIPTION OF THE INVENTION

Through the present invention, specific polypeptide molecules areobtained, formed by one or more antigen binding sites, coming from amouse Mab that is specific for human CEA. The antigen binding site isassembled in the form of monovalent, divalent, and other forms ofantibody fragments, depending on the way the polypeptide molecule isconstructed.

The polypeptide molecule in the form of a monovalent scFv fragmentspecific for human CEA exhibits an affinity constant for this antigen of(5.0±0.4)×10⁹ L mol⁻¹, and comprises the VH and VL domains, linked inthis order by a 14-aminoacid union segment (linker), with an aminoacidsequence as the one presented in SEQ ID No. 16.

The polypeptide molecule in the form of a divalent scFv fragment(diabody) specific for human CEA exhibits an affinity constant for thisantigen of (2.8±0.3)×10¹⁰ L mol⁻¹, and comprises the pairing of twoidentical molecules formed each one by the VH and VL domains, linked inthis order by a five-aminoacid union segment (linker), with an aminoacidsequence as the one presented in SEQ ID No. 17.

In another aspect of the invention, the monovalent and diabody scFvfragments do not bind, or bind in a non significant manner, with normaltissues, or cells from the following normal tissues: liver, kidney,lung, testicle, blood, spleen, and pancreas. In the case of the colonmucosa, the monovalent and diabody scFv fragments react exclusively withthe products of luminal secretion and in apical zones or some glands.The absence of reactivity of the monovalent and diabody scFv fragmentswith normal lymphocytes and neutrophils is indicative that there is notan important level of cross reactivity with the NCA antigen (von KleistS, Burtin P. Immunodiagnosis of Cancer. Marcel Dekker. 322-341, 1979;Buchegger, F. et al. Int. J. Cancer 33; 643-649, 1984).

The monovalent and diabody scFv fragments can bind to soluble CEA, CEAabsorbed to solid surfaces, or CEA associated to cells that produce it,and to tumor tissues, among which human colorectal, breast, lung,pancreas and stomach adenocarcinomas stand out. The monovalent anddiabody scFv fragments and the Mab CB/ior-CEA.1 bind to soluble andsolid surface bound CEA in a form that is dependent of the conservationof glycosylation of human CEA, suggesting that the carbohydrates of thisantigen are involved in the recognition.

Polypeptide molecules derived from the monovalent and diabody scFvfragments reported in this invention, that retain the capacity ofbinding CEA, their reported affinity, specific epitope recognition, andsimilar and equivalent biological and biochemical performance to thefragments described in this invention are considered equivalent variantforms and are contained in the present invention. These polypeptidemolecules can take the form of other recombinant antibody fragments,such as scFv where the VL domain precedes the VH, or Fab, Fab′, F(ab′)2,Fabc, Facb, trimeric and tetrameric scFv, etc. (Winter G, Milstein C.Nature 349: 293-299, 1991; WO94/13804; de Haard, H et al. Adv. DrugDelivery Rev. 31:5-31, 1998), and other union segments (linkers) knownin the state of the art are used. These can also be in the form ofbi-specific antibody molecules, where a portion of such conservespecificity for CEA, and the other has a different specificity.

Equally contained in the present invention are the variant forms of themonovalent and diabody scFv fragments that comply with thecharacteristics described in the previous paragraphs, and that wouldhave been derived from the so-called “humanization by immunogenicityreduction”, in which B and T cell epitopes present in variable domainsare modified in a way that the antigenic recognition is not altered, butthe immunogenicity of the resulting molecule in humans is reduced, forexample, as it is revealed in Carr F J et al. 2000 EP 983303A1 and inRodriguez Perez R et al. U.S. Pat. No. 5,712,120-A. Equally consideredvariant forms contained in this invention are those produced by theso-called “CDR transplant” in which the CDR sequences of a firstantibody are placed within the frame of sequences that are not from thisantibody, for example, as it is revealed in EP-B-0239400, EP-A-184187,GB 2188638A or EP-A-239400, and retain the capacity of binding CEA withsimilar affinity, competitive capacity, particular epitope recognition,and biological and biochemical performance similar and equivalent to themonovalent and diabody scFv fragments described in this invention.

Apart from the antibody sequences, the polypeptide molecules containedin this invention can comprise other aminoacids that form a peptide orpolypeptide, or that add to the molecule a functional characteristicdifferent to that of binding the CEA antigen, as for example a tag forpurification or identification, an enzyme or its fragments, a biologicalresponse modifier, a toxin or drug, and successively.

In agreement with this invention the monovalent and diabody scFvfragments can be administered in isolated or purified form.

The present invention foresees the use of some of the polypeptidemolecules described above as a diagnostic reagent for human cancer formsthat express CEA, as for example, colon, lung, breast or otheradenocarcinomas.

The polypeptide molecules specific for CEA describe above can beradiolabeled and employed as agents to obtain images to demonstrate in aspecific way the presence and location of tumors that express CEA inhumans. The present invention provides a method to determine thepresence of a cell or tumor that express CEA, being such method that ofplacing in contact the cells with a polypeptide molecules as the onesdescribed, and determining the binding of these to the cells. The methodcan be developed in vivo, or in a sample of cells removed from the body,being this in vitro or ex vivo.

The present invention provides a method fro the binding of a polypeptidemolecule as the ones described before, to human CEA. This binding canhappen in vitro, ex vivo or in vivo. If the binding is in vivo, themethod can comprise the administration of the polypeptide molecule tomammals, being these one or several individuals. As t is demonstratedexperimentally here, the monovalent and diabody scFv fragments in thisinvention bind to human CEA expressed by transfected mouse tumor cells,which grow as tumors once they are transplanted to mice, providing anexperimental model useful for the study, the investigation, and thedevelopment of molecules with specific binding and of their properties.

The reactivity of the antibodies on cellular samples can be detectedthrough any appropriate mean. Labeling with individual reportermolecules is one such possibility. Reporter molecules can generatesignals capable of being detected directly or indirectly and preferablymeasured. The coupling of the reporter molecules can be direct orindirect, covalent or non covalent. The union through a peptide bond canresult from the recombinant expression of a gene fusion that couples theantibody and the reporter molecule. The form of determining the couplingis not a characteristic of the present invention, and those skilled inthe art are capable of choosing an adequate model in accordance to theirpreference and general knowledge.

When a radionuclide as ¹²⁵I, ¹¹¹In or ^(99m)Tc is used to label themonovalent and diabody scFv fragments and its equivalent forms, if theselocate preferably in the tumor, and not in the normal tissues, thepresence of he radioactive labeling in the tumor tissue can be detectedand quantitated using a gamma camera. The quality of the obtained imageof the tumor correlates directly with the ratio signal:background(Goldenberg D M. Int. J. of Biol. Markers 1992, 7; 183-188). Theexperimental use of ¹²⁵I is exemplified in the text.

The present invention also offers elements so that the monovalent anddiabody scFv fragments and their equivalent variant forms as describedbefore, can be used as a therapeutic reagent, fro example, when they arecoupled, conjugated or bound to molecules with therapeutic power, or aregenerated as a recombinant fusion protein. The monovalent and diabodyscFv fragments and their equivalent variant forms according to thepresent invention can be used to direct a toxin, radioactivity, T and NKcells, or other molecules to tumors that express CEA, or to develop ananti-idiotypic response in the organism that could conduct to a desiredtherapeutic effect. In agreement with this, other aspects of theinvention provide elements for methods of treatment that involve theadministration of monovalent and diabody scFv fragments or theirequivalent variant forms, as medicaments or pharmaceutical compositions.

In agreement with the present invention, the compositions can beadministered to individuals, preferably in a “therapeutically effective”amount, sufficient to demonstrate a benefit for the patient, in the wayof the improvement of at least one symptom. Details related to theamount to administer, the frequency and intervals of administration willdepend on the nature and severity of the disease that is treated, andthese decisions are the responsibility of specialists and other medicaldoctors. The appropriate doses of an antibody are well known in the art(Ledermann J. A. et al. Int J. Cancer 47: 659-664, 1991; Bagshawe K D etal. Antibody, Immunoconjugates, and Radiopharmaceuticals 4: 915-922,1991).

A composition can be administered alone or in combination with othertreatments, either simultaneously or sequentially, depending of thedisease to be treated.

The pharmaceutical compositions in agreement to the present invention,and to be employed according to the present invention, can comprise,apart from the active ingredient, an excipient, buffer, stabilizer oraccepted pharmaceutical carrier, or other materials well known for thoseskilled in the art. These materials should not be toxic, should notinterfere with the efficacy of the active ingredient and their precisenature could depend on the administration route, being this oral, or byinjection, for example, intravenously.

The scFv monovalent and diabody fragments and their equivalent variantforms in agreement with the present invention can be fabricated throughthe expression of the encoding nuclei acid. The nucleic acid thatencodes for any of these polypeptide molecules described before is partof the present invention, as it is a method for the expression of suchnucleic acid. In a different embodiment, the nucleic aid can encode forthe aminoacid sequences shown in SEQ ID No. 16 and 17.

For the recombinant expression of the monovalent and diabody scFv andtheir equivalent variant forms, appropriate vectors can be selected orconstructed, with the adequate regulatory sequences, including promoter,terminator, enhancer, polyadenilation, marker genes, and other pertinentsequences. The vectors can be plasmids. Many known protocols andtechniques for the manipulation of nucleic acids, for example,preparation of nuclei acid constructions, polymerase chain reaction,mutagenesis, sequencing, introduction of DNA in cells and geneexpression, protein analysis, and others are described in detail inseveral references, as Molecular Cloning: a Laboratory Manual: 2ndedition, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989 orShort Protocols in Molecular Biology, Second Edition, Ausubel et al.eds., John Wiley & Sons, 1992 or Erlich H A PCR Technology, StocktonPress, 1989. The revelations that appear in these references areincorporated in this document by referral.

Another aspect of the present invention provides a host cell containinga foreign nuclei acid and the methods to introduce such nucleic acid ina host cells. The introduction can employ any of the techniques thatexist for such purpose. For bacterial and yeast cells, this techniquecan be the electroporation. The introduction can be followed byprovoking or allowing the expression of the nucleic acid, for example,growing the host cells under conditions favorable for the expression ofthe gene. In one embodiment, the nucleic acid of the inventionintegrates in the genome of the host cells.

After their production, the monovalent and diabody scFv fragments andtheir equivalent variant forms, can be used in any of the forms revealedhere, such as in the formulation of a composition as a pharmaceutical,or a diagnostic product, such as in a set of reagents that comprisesapart from the specific binding member, one or more reagents todetermine the binding of the member to cells or to CEA not linked tocells, as discussed before.

Other further aspects of this invention and its realizations will beapparent to those experts in the art. For the complete understanding ofthis invention, and not to limit its extension and reach, examples areprovided. Reference is made to the following figures:

SHORT DESCRIPTION OF THE FIGURES

In FIG. 1, a scheme of the pJG-1m vector used for the expression of themonovalent scFv and the diabody in E. coli is represented. Incorrespondence with the vector zone marked with the wide horizontal bar,the base sequences of the fragments cloning site, the c-myc peptide, the6-hitidine domain, and some inter and pos-domain regions are presented(SEQ ID No. 13).

In FIG. 2 the alignment of the aminoacid sequences (in one-letter code)deducted from the nucleotide ones for (1) the monovalent scFv fragment(SEQ ID No. 16), and (2) the divalent fragment (diabody) (SEQ ID No.17), are presented. The order of the domains in both constructions areVH-linker segment-VL. The aminoacids of the linker segments employed ineach of the two molecules appear in bold characters.

In FIG. 3 the recognition of (A) Mab CB/ior-CEA.1, (B) monovalent scFv,and (C) diabody, for the CEA expressed in culture tumor cells AsPC-1(ATCC CRL-1682) is exemplified, through the indirect immunofluorescencetechnique. In A, B, and C, the characteristic membrane and nearbycytoplasm fluorescence is seen. Magnification is 200×.

In FIG. 4 the chromatographic profile of the proteolytic digestion ofthe diabody and the assignation of tryptic peptides obtained by massspectrometry is presented. Up: Chromatographic profile of the trypticdigestion of the diabody. Down: Summary table of the assignation of thediabody tryptic peptides. M/z exp: experimental mass; theorical m/z:theoretical mass; Z: charge. In the obtained spectra signalscorresponding to incorrectly linker cysteines were not detected.

FIG. 5 is a summary of the aminoacids sequence verification of thediabody (SEQ ID No. 21). The regions of the protein sequence that wereverified by mass spectrometry are outlined in bold characters, and thezones of the sequence that were not recovered after tryptic digestionappear in italics. The zones in bold coincide in total with theaminoacid sequence deducted from the base sequence of the diabody. Thesequence of the c-myc peptide and the final 6 histidines, provided bythe pJG-1 m vector (FIG. 1), are also seen in the C-terminus portion.

FIG. 6 presents the percentage of the injected dose per gram of tissue,after 24 (stripe bars) and 48 (non-striped bars) hours of inoculation ofmice bearing tumors that express human CEA with the following moleculesradiolabeled with ¹²⁵I: from left to right, and in groups of four doublebars: (a) diabody, (b) scFv, (c) F3, and (d) Mab CB/ior-CEA.1. Each barrepresents the mean of the counts recovered from the organs obtainedfrom 12 mice. The results demonstrate that between 24 and 48 hours, theratio radioactivity in tumor: radioactivity in blood is maintained highfor the diabody, the scFv, and Mab, with the highest values for thelatter, followed by the dimeric molecule. F3 showed very low values,with an in vivo behavior inadequate that can be correlated by itsdiminished affinity for CEA.

All documents mentioned herein are incorporated by reference.

EXAMPLES

-   -   1. Amplification by PCR, cloning, and sequencing of the variable        domains of Mab CB/ior-CEA.1    -   2. Assembly of scFv and diabody, expression in E. coli, and        demonstration of their recognition of human CEA    -   3. Expression of the scFv and diabody in Pichia pastoris and        demonstration of their recognition of human CEA    -   4. Purification of the scFv and diabody produced in bacteria    -   5. Characterization of the diabody through proteolytic digestion        and mass spectrometry.    -   6. Studies of recognition of deglycosylated CEA    -   7. Immunocyto- and histo-chemical study in normal and tumor        tissues.    -   8. Determination of the affinity constant.    -   9. Determination of the specific recognition in vivo of        fragments and antibody labeled with ¹²⁵I, in C57BI/6 mice        bearing tumors induced by the inoculation of B16-CEA13 cells.

Example 1 Amplification by PCR, Cloning, and Sequencing of the VariableDomains of the Mab CB/ior-CEA.1

Procedure (a) Purification of RNA and Amplification of Variable Regions

Total RNA from 10⁶ cells of the mouse hybridoma CB/ior-CEA.1 (Tormo B.et al. APMIS. 97: 1073-1080, 1989) was extracted with the TriPure™reagent (Boehringer-Mannheim). The complementary DNA (cDNA) wassynthesized using the First-Strand cDNA Synthesis for RT-PCR Kit(Boehringer-Mannheim), using oligo dT as primer. The polymerase chainreaction technique (PCR) for the specific amplification of the heavy andlight chain variable domain genes was used. The employed syntheticprimers were designed on the basis of the consensus sequences for mouseIgG and kappa chains, reported by Kabat E. et al. (US Department ofHealth and Human Services, NIH, 1991) and experiments developedpreviously in this laboratory (Coloma, M J et al. Biotechniques 11:152-156, 1991). The sequences of the oligonucleotides used in the PCRappear in Table I. TABLE I Synthetic oligonucleotides used in the PCRfor the amplification of the sequences that encode for the heavy (VH)and light (VL) chain variable domains of he Mab. CB/ior-CEA.1. HeavyChain Oligo 1. Signal Peptide of VH. (SEQ ID No. 1) 5′ . . .GGGGATATCCACCATGRACTTCGGGYTGAGCTKGGTTTT . . . 3′ Oligo 2. CH1 region(SEQ ID No. 2) 5′ . . . AYCTCCACACACAGGRCCAGTGGATAGAC . . . 3′ LightChain Oligo 3. Signal Peptide of VL. (SEQ ID No. 3) 5′ . . .GGGGATATCCACCATGGAGWCACAKWCTCAGGTCTTTRTA . . . 3′ Oligo 4. Ck constantregion (SEQ ID No. 4) 5′ . . . ACTGGATGGTGGGAAGATGGA . . . 3′

For PCR, the PCR Core Kit (Boehringer-Mannheim) was used. The conditionsof the PCR were: denaturizing to 94° C., 1 minute, annealing to 55° C.,1 minute, extension to 72° C., 1 minute, 25 cycles, with 5 additionalminutes of extension to the temperature already described in the lastcycle, everything in a MJ Research Minicycler equipment. The finalvolumes of each reaction were 100 μL. All the oligonucleotides were usedto a final concentration of 1 μM.

The DNA amplified fragments, with the expected size of around 320-530bp, were purified in low meeting point agarose gels (Sigma), using theQIAquick Gel Extraction Kit (QIAGEN, GmbH), and were clonedindependently in the pMOS vector (Amersham Pharmacia Biotech), designedfor the “blunt” cloning of DNA fragments.

Procedure (b). Nucleotide Sequence of the Variable Domains

For the determination of the nucleotide sequence of the light and heavychain variables domains cloned in the vector pMOS, the oligonucleotidesrecommended by the manufacturer were used (Amersham Pharmacia Biotech).The base sequence was made by means of automatic methods, using anALFexpress II equipment of Pharmacia (Amersham Biosciences), and the“Thermus Sequenase 5 Cy Dye Terminator Kit”. The plasmids pVL2 and pVH5were selected as representative of the sequences of VL and VH,respectively.

Example 2 Assembly of the ScFv and Diabody, Expression in E. coli andDemonstration of Its Recognition of Human CEA

Procedure (a). Re-amplification of the Variable Domains and Assembly ofScFv and Diabody

The PCR was used for the assembly, in the form of scFv and diabody, ofthe VH and VL domains contained in the plasmids pVH5 and pVL2.

The synthetic oligonucleotides were designed on the basis of thesequences of VH and VL in the plasmids pVH5 and pVL2. These includedsites of restriction for the cloning in the vector pJG-1m andincorporated the linker segments of 14 and 5 aminoacids for the assemblyof monomeric scFv and diabody (Tables II and III). TABLE III Syntheticoligonucleotides used in the PCR for the assembly of scFv and diabody.Oligo 5. ApaL1- FR1 VH (SEQ ID No. 7) 5′ . . .TCTCACAGTGCACAGGAAGTGAAGCTGGTGG AGTCTGGG . . . 3′ Oligo 6. Linker of 14aminoacids /FR4 VH (SEQ ID No. 8) 5′ . . .GTCGACTTTGGATTCGGAGCCTGATCCTGAGG ATTTACCCTCTGAGGAGACTGTGAGAGTGGT . . .3′ Oligo 7. Linker of 14 aminoacids/FR1 VL (SEQ ID No. 9) 5′ . . .GAGGGTAAATCCTCAGGATCAGGCTCCGAAT CCAAAGTCGACGACATTGTGATGACCCAGTC . . . 3′Oligo 8. Not I- FR4 VL (SEQ ID No. 10) 5′ . . .AAGGAAAAAAGCGGCCGCTTTCAGCTCCAGC TTGGTT . . . 3′ Oligo 9. Linker of 5aminoacids /FR4 VH (SEQ ID No. 11) 5′ . . .AGAGCCGCCGCCACCTGAGGAGACTGTGAGA GTGGT . . . 3′ Oligo 10. Linker of 5aminoacids/FR1 VL (SEQ ID No. 12) 5′ . . .GGTGGCGGCGGCTCTGACATTGTGATGACCC AGTCT . . . 3′

For the assembly of fragments, independent PCR were made in a first stepto amplify:

-   -   1. For the domains that would give origin to the monovalent        scFv.—Reaction 1: using the plasmid pVH5 as template with        oligonucleotides 5 and 6 (Table III). Reaction 2: using the        plasmid pVL2 with oligonucleotides 7 and 8 (Table III).    -   2. For the domains that would give origin to the        diabody.—Reaction 3: using the plasmid pVH5 with        oligonucleotides 5 and 9 (Table III). Reaction 4: using the        plasmid pVL2 as template with oligonucleotides 8 and 10 (Table        III).

The conditions and reagents used for the PCR were already describedabove. All the oligonucleotides were used to final concentration of 1μM.

For the assembly of scFv a new PCR was made mixing 4 μL of reactions 1and 2 with oligonucleotides 5 and 8 (Table III) in final concentrationof 1 μM, and oligonucleotides 6 and 7 (Table III) in final concentrationof 0.01 μM.

For the assembly of the diabody a new PCR was made mixing 4 μL ofreactions 3 and 4 with oligonucleotides 5 and 8 (Table III) in finalconcentration of 1 μM, and oligonucleotides 6 and 7 (Table III) in finalconcentration of 0.01 μM.

The amplified DNA fragments were detected as majoritary bands ofapproximately 700 bp, and were isolated from low melting point agarosegels, as was described previously.

Procedure (b). Cloning in pJG-1m Vector.

The vector pJG-1m is a plasmid designed for the expression of antibodyfragments in the periplasm of E. coli (FIG. 1). As main elements it hasthe LacZ promoter, a signal peptide, restriction sites ApaL I and Not Ifor the insertion of the fragment gene, a c-myc peptide and a sequencethat encodes for 6 histidines. This last one is used as tag for thepurification of expression products by immobilized metal ions affinitychromatography (IMAC; Porath J. Prot. Expr. Purif. 3: 263-281, 1992).The base sequences of the cloning restriction sites for the cloning ofthe scFv in question in the vector, and of the C-terminus amino acidsthat are added to the scFv, appear in FIG. 1 (SEQ ID No. 13).

The DNA fragments corresponding to scFv and diabody, and the pJG-1mvector were digested with ApaLI and Not I (Promega) restriction enzymesand the bands and vector ligated independently using T4 DNA ligase(Promega). The products of the ligation reactions were used for thetransformation of competent E. coli (XL-1Blue strain; Stratagene) byelectroporation, and the transformed cells were grown in solid selectivemedium (LB agar, with 100 μg/mL of ampicillin) during 16 hours at 37° C.The used methods are described in Molecular Cloning, A LaboratoryManual, Second Edition. Sambrook, Fritsch, Maniatis. 1989.

The recombinant plasmids were selected after the purification of theplasmid DNA from several colonies (QIAGEN MiniPrep kit), and thecorresponding checking by digestion with the restriction enzymes alreadydescribed for expected ligation products. In the restriction analyses,bands of approximately 3.5 kb were obtained corresponding to thelinearized vector, and bands of approximately 700 bp for the genesencoding the scFv and diabody antibody fragments.

Five clones of each construction were sequenced using specificallydesigned primers that hybridize externally to the cloning regions of thevector pJG-1m (Table IV), by means of previously described procedures.TABLE IV Synthetic oligonucleotides for the bases sequencing of the scFvand diabody assembled by PCR and cloned in vector pJG-1m. Oligo 11. (SEQID No. 14) 5′ . . . GTTGTTCCTTTCTATTCTCAC . . . 3′ Oligo 12. (SEQ ID No.15) 5′ . . . CTCTTCTGAGATGAGTTTTTGTTC . . . 3′

The aminoacid sequences derived from the base sequences obtained for themonovalent scFv (clone pJG1m-25) and the diabody (clone pJG1m-18) appearin FIG. 2 (SEQ ID No. 16 and SEQ ID No. 17). With respect to a scFvdeveloped previously (Ayala M et al. Biotechniques 13: 790-799, 1992),the VH and VL sequences now obtained for the new monovalent scFv anddiabody exhibit 16 different aminoacids within the VH FR1, CDR2 and FR3domains, and 3 different aminoacids within the VL FR1 and FR3 domains.

These results indicate that the variable domains amplified and clonedfrom hybridoma CB/ior-CEA.1 to construct the new monovalent scFv anddiabody can come from RNA different with respect to the ones used in theamplifications for the clonings of the previously reported scFv.

The linker segment of the new monovalent scFv is identical to the one ofthe scFv reported previously. The segment of union of the new scFvdivalent (diabody) is different from the one of the scFv obtainedpreviously, because it only includes 5 amino acids. In these experimentsthe sequences of the linker segments L1 and L2 were also verified, thatappear in Table II.

Procedure (c) Verification of the Expression in E. coli of the ScFv andDiabody by SDS-PAGE and Western blot.

Competent E. coli cells TG1 were transformed independently with plasmidspJG1m-25 and pJG1m-18, containing the information for both antibodyfragments. This strain allows the periplasmatic expression of theheterologous protein, or its secretion towards the culture medium.

The transformed bacteria were plated on solid selective medium and grownat 37° C. for 16 hours. A representative colony of each of the twoconstructions were grown on liquid medium until OD_(530 nm)=1 andinduced for 12 hours adding 1 mM of IPTG to the culture medium. Thecells were centrifuged and the periplasmatic content isolated by osmoticshock and brief sonication (seconds) for its evaluation inelectrophoresis in 12% SDS-polyacrilamide gels (SDS-PAGE). This testrevealed the expression in both cases of proteins of the expectedmolecular size (approximately 27 kDa), that were later evaluated byWestern Blot using as primary antibody a Mab (9E10) specific against thepeptide derived from c-myc that this protein contains (1 μg/mL),followed by rabbit anti-mouse IgG antibodies conjugated with horseradishperoxidase (Sigma). The transference of proteins from the SDS-PAGE toHybond C Extra nitrocellulose (Amersham Life Sciences) was done in asemi-dry transference equipment (BioRad). DAB (Sigma) insolublesubstrate was used in the development.

For the two constructions, the recombinant proteins with the mentionedsize were identified with the Mab 9E10.

Procedure (d) Specific Recognition of the Human CEA by ScFv and Diabodyby ELISA

An ELISA test was made coating polyvinyl plates (Costar, 96-well VinylAssay Plates) with human CEA (Calbochem 219369), to a concentration of 1μg/mL. After blocking the plates with skim milk, the bacterial periplasmsamples corresponding to the two constructions were added in dilutionsof 1:5, 1:10, and 1:20 in PBS-2% skim milk, and incubated by 2 hours atroom temperature.

For the detection of the union of fragments to the CEA, Mab 9E10 (1μg/mL) was used, followed by mouse anti mouse IgG antibodies conjugatedwith horseradish peroxidase from Sigma. After several washings, OPD(Sigma) and H₂0₂ as chromogen and substrate were used to developl thereactions, and the quantitative evaluation of the reactions read at 492nm in a LabSystems Multiskan MS.

In the test, Mab CB/ior-CEA.1 was used as positive control. Periplasmfractions corresponding to cells TG1 transformed with the vector pJG-1mwithout insert, and a non-related Mab, were used as negative controls.Also, plates were coated with the following irrelevant antigens: 10μg/mL of bovine seroalbumine (BSA, Sigma), 10 μg/mL of ovalbumine, 10μg/mL of lyzozyme, 10 μg/mL of keyhole limpet haemocyanin (Sigma). Inall the plates wells were included where only phosphate buffered salinesolution (PBS) without antigen (blank) was placed. Values of absorbanceat least 4 times greater than the produced by the negative controls wereconsidered positive.

In these experiments the samples of periplasm of the constructions ofscFv and diabody resulted positive with respect to their capacity ofrecognition of human CEA adsorbed to polyvinyl plates. These samesamples were negative for all the irrelevant antigens.

Procedure (e) Recognition of Human CEA Associated to Cells by the ScFvand Diabody in ELISA and Indirect Immunofluorescence

The human tumor cell lines LoVo (ATCC CCL-229), AsPC-1 (ATCC CRL-1682),and LS 174T (ATCC CL-188), all which expresses CEA in culture, wereseeded in 96 wells polystyrene plates (Costar). Once the confluence wasreached, the wells were washed twice with PBS, drained off, andair-dried. The cells were then fixed to the plastic by using a 1:1 (v:v)mixture of cold acetone-methanol, for 3 minutes. After several washingswith distilled water to eliminate residues, the plates were used assolid phase in ELISA tests where the samples of bacterial periplasmcorresponding to the two constructions were added in dilutions of 1:2,1:8, and 1:16, in PBS-2% skim milk, and incubated for 2 hours at roomtemperature. After several washings, Mab 9E10 (1 μg/mL) was used,followed by anti mouse IgG antibodies conjugated with horseradishperoxidase (Sigma) for the detection of the union of fragments to CEA.After several washings, the chromogen OPD (Sigma) and H₂0₂ as substratewere used to develop the reactions, and a LabSystems Multiskan MS readeremployed for quantitative evaluation of the reactions at 492 nm. For thereading step, the supernatants were transferred to a fresh plate. TheMab CB/ior-CEA.1 was used as positive control in the assay. Periplasmfractions corresponding to cells TG1 transformed with the vector pJG-1mwithout insert, and a non-related Mab, were used as negative controls. Aplate with human cells HEK 293 (ATCC CRL-1573), that do not express CEA,was also used as negative control. The criteria of positivity weresimilar to the ones used in the ELISA described in the previousProcedure.

In this experiment the periplasm samples of the scFv and diabodyconstructions only recognized LoVo, AsPC-1 and LS 174T cells. All thenegative controls were negative. In this way, the capacity of the scFvand diabody to identify the human CEA on human tumor cells that expressthis antigen, fixed on polystyrene plates, by cell-ELISA, wasdemonstrated.

In another experiment, LoVo, AsPC-1 and LS 174T cells were seeded in 35mm diameter polystyrene plates (COSTAR) and cultivated until theconfluence was reached. The plates were washed twice with PBS, drainedoff, they air-dried, and the cells fixed to the plastic using a 1:1(v:v) mixture of cold acetone-methanol. After several washings withdistilled water to eliminate residues, the plates were used as solidphase in indirect immunofluorescence tests. For this, circular zoneswere defined in the surface with fixed cells, in which bacterialperiplasm samples corresponding to the two constructions, in dilutionsof 1:2, 1:4 and 1:8 with PBS-3% BSA, were incubated independently. Thesame positive and negative controls used in the cell-ELISA wereemployed.

The incubation was at room temperature (RT) for 1 hour in humid chamber,followed by several washings with cold PBS-3% BSA, and the addition ofMab 9E10 (10 μg/mL) to all the monolayer by one hour at RT, also inhumid chamber. After several washings with cold PBS-3% BSA, themonolayer was incubated with anti mouse IgG antibodies conjugated withfluorescein isothiocyanate (FITC, Sigma) diluted 1:64 in PBS-3% BSA, for30 minutes, in the dark and humid chamber, then were washed five timeswith PBS-3% BSA, once more with PBS, and finally stained with Evans Bluesolution for a few minutes.

The monolayer was covered with PBS-10% glycerol, sealed with a coverslip and examined with a fluorescent light accessory Olympus BH2-RFL,mounted in an Olympus BHT microscope. Plates with HEK 293 human cellswere also used as negative control. The presence of membrane andcytoplasm apple-green fluorescence was established as criterion ofpositive result, as long as this would not exist in the negative controlsamples, or in the human cells negative for CEA.

In this experiment, the periplasm simples of the scFv and diabodyconstructions only recognized the LoVo, AsPC-1 and LS 174T cells. Thenegative controls were negative. In this way, the capacity of the scFvand diabody to identify the human CEA on human tumor cells that expressthis antigen, fixed on polystyrene plates, by indirectimmunofluorescence, was demonstrated. An example of the results is shownin FIG. 3.

Example 3 Expression of ScFv and Diabody in Pichia pastoris andDemonstration of Its Recognition of Human CEA

Procedure (a) Re-amplification of ScFv and Diabody and Cloning in theVector pPS7.

The genes that codify for the scFv and diabody were amplified by PCRusing as templates the constructions pJG1-25 and pJG1-18, respectively,and oligonucleotides designed to add the NcoI site in the 5′ and 3′ endsof the genes (Oligos 13 and 14; Table V), with the purpose of cloning inthe Pichia pastoris expression vector pPS7. The amplification procedurewas similar to the one described previously. Plasmid pPS7 is anintegrative vector that contains a fragment of 1.15 Kb that correspondsto the promoter of the alcohol oxidase (AOX.1) enzyme followed by thegene that codifies for the secretion signal of the sucrose invertase(sucII) of Saccharamyces cerevisae, a unique NcoI cloning site, afragment of 960 bp of the enzyme glyceraldehyde 3-phosphatedehydrogenase (Gapt) to guarantee the completion of the transcription,and the HIS3 gene of Saccharamyces cerevisae as selection marker. Inaddition this vector contains a fragment of 2.1 kb, corresponding to the3′ sequence of the AOX.1gene. All these elements are inserted in avector pUC18 (Herrera Martinez L S et al., EP0438200 A1). TABLE VSynthetic oligonucleotides employed in the PCR for the amplification andmodification of the sequences that encode for the first bases of VH andthe last of VL, for the cloning of the scFv and diabody in the pPS7vector, and in the sequencing of these clonings. Oligo 13. Nco 1 - FR1VH (SEQ ID No. 18) 5′ . . . CATGCCATGGGGAATCCGAAGTGAAGCTGGTGGAG . . . 3′Oligo 14. Nco 1 - 6 histidines (antisense) (SEQ ID No. 19) 5′ . . .CATGCCATGGATCCCGGGGTGATGGTGATGGTGATG . . . 3′ Oligo 15. alcohol oxidasepAOX.1 promoter (SEQ ID No. 20) 5′ . . . GACTGGTTCCAATTGACAAGC . . . 3′

After the NcoI digestion (Promega) of the amplified bands correspondingto the scFv and diabody, these were ligated independently to the vectorpPS7 previously digested with the same enzyme, and the products of theligation were used to transform in an independent way the XL-1Bluestrain of E. coli. Isolated colonies corresponding to the transformationof the strain with each recombinant vector were analyzed using colonyPCR with a primer that hybridizes in the promoter (Oligo 15, Table V)and another for the 3′end of VL (Oligo 8, Table III). Colonies thatcontain the correct insert oriented were selected. The sequencing of thecloned genes was made according to the previously described procedure(EXAMPLE 1 Procedure b), using Oligo 15 (Table V). The sequencesobtained for the VH and VL domains of the recombinant plasmids pPSM2(scFv) and pPSM3 (diabody) agreed with the previously cited in SEQ IDNo. 16 and SEQ No. 17.

Recombinant strains of Pichia pastoris were obtained with these twoplasmids through the electroporation of the MP36 his 3 wild strain (YongV ET to. Biotechnol. Applic. 9: 55-61, 1992) with both mentionedplasmids, previously digested with the restriction enzyme PvuII(Promega), and selecting on histidine deficient minimum medium. As aresult of the different recombination mechanisms of the recombinantplasmids with specific sites in the genome of Pichia pastoris, it waspossible to isolate for each construction two different types ofphenotypes of secretory strains: (a) strains in which the AOX.1 gene wasnot affected during the recombination event and therefore grew in mediawith methanol and showed to growth similar to the wild strain (Mut+),and (b) strains in which the AOX.1 gene was replaced by the expressioncassette and showed slow growth in the presence of methanol (Mut s).

Procedure (b) Expression Studies

The studies of antibody fragment expression were made starting from theprototrophic colonies His+ grown in plates with selective MD medium(nitrogen yeast base, biotin, dextrose). The selected colonies wereinoculated in 10 mL of rich BMGY buffered medium (yeast extract,peptone, potassium phosphate, nitrogen yeast base, biotin, and glycerol)in 50 mL tubes, and were placed at 28° C. with rotation at 150 rpm. Whenthe cultures reached 2 units of OD 600 nm, measured in a SPECTRONICGENESIS 2 equipment, these were centrifuged at 2000 rpm, during 10minutes. The cellular pellets were suspended in 10 mL of rich mediumwith methanol (BMMY) as unique carbon source, instead of glycerol. Fromthis moment on and during 96 hours the proteins of interest wereinduced, with daily addition of pure methanol until a finalconcentration of 1% in the culture. As negative control the MP36his3strain transformed with a vector without insert was used.

Finalized the period of culture, the cells were centrifuged, the culturemedium metabolized during the phase of induction collected, centrifugedonce again for its final clarification and detection of scFv or diabodydone by electrophoresis in 15% gels of SDS-polyacrilamide (SDS-PAGE).This test revealed the expression of proteins of the expected molecularweight in both cases (approx 27 kDa), that were later evaluated byWestern Blot using the Mab 9E10 as primary antibody, and rabbitanti-mouse IgG antibodies conjugated with horseradish peroxidase (Sigma)as secondary antibody. The transferences were made as described above.In the development, DAB (Sigma) was used as insoluble substrate. For thetwo constructions, the recombinant proteins were identified with the Mab9E10.

Procedure (c) Recognition of the Human CEA by ScFv and Diabody in ELISA.

An ELISA test, very similar to that previously described for thematerial derived from E. coli, was made using similar solid phases,reagents and coating, incubation, development, and positive controlconditions. The samples of metabolized culture of the inducedrecombinant strains were diluted in PBS-1% milk and added at the rate of100 μL/well, and incubated for 2 hours at room temperature. As negativecontrols, metabolized medium corresponding to the strain MP36his 3, anda unrelated Mab, were used. Values were considered positives whenabsorbance was at least 4 times greater than that produced by thenegative controls.

In this experiment the samples of induction-phase metabolized medium ofthe scFv and diabody constructions expressed in Pichia pastoris werepositive with regards to their recognition capacity of human CEAadsorbed to polyvinyl plates.

Procedure (d) Recognition of the Human CEA Associated to Cells byCell-ELISA and Indirect Immunofluorescence.

An ELISA test, very similar to that previously described for thematerial derived from E. coli, was made using similar solid phases,reagents and coating, incubation, development, and positive controlconditions. The samples of metabolized culture of the inducedrecombinant strains were diluted in PBS-2% milk and added to the plateswith fixed LoVo, AsPC-1, and LS 174T cells, and incubated for 2 hours atroom temperature with gentle stirring. In the test, Mab CB/ior-CEA.1 wasused as positive control. Metabolized induction phase cultures of strainMP36his transformed with the vector pPS7 without insert, and anunrelated Mab, were used as negative controls. Also, as negativecontrol, a plate with human HEK 293 cells was used.

In this experiment the capacity of the scFv and diabody for specificidentification of human CEA on human tumor cells fixed on polystyrenesupports, by ELISA, was demonstrated.

An indirect immunofluorescence test, very similar to the previouslydescribed for the material derived from E. coli, was used, employingsimilar culture cells, and conditions of fixation, reagents, incubation,development, mounting, microscope observation and positive criterion.Independent zones were defined to the plates with fixed LoVo, AsPC-1 andLS 174T cells, in which the samples of induced cultures of therecombinant stocks corresponding to the two constructions, and negativeones, diluted in PBS-3% BSA, 0.02% sodium azide were applied.

The incubation was done at room temperature (RT) for 1 hour in humidchamber, followed of several washings with cold PBS-BSA-sodium azide,and the addition of Mab 9E10 to all the monolayer for 1 hour at RT, alsoin humid chamber. After several washings with cold PBS-3% BSA, themonolayer was incubated with anti mouse IgG antibodies conjugated withfluorescein isothiocyanate (Sigma) diluted 1:64 in PBS-3% BSA for 30minutes in the dark and humid chamber. The plates were washed five timeswith PBS 3% BSA, once with PBS, and finally stained with Evans Bluesolution for a few minutes. The monolayers covered with PBS-10% glycerolwere mounted with coverslips and examined in the ultraviolet lightmicroscope.

In the assay, Mab CB/ior-CEA.1 was used as positive control. As negativecontrols the induced culture medium of MP36his3 transformed with pPS7without insert, and a unrelated Mab, were used. Also a slide with HEK293 cells was used as negative control. In this experiment the samplesof the induced cultures that secreted the recombinant scFv and diabodyrecognized only LoVo, AsPC-1 and LS 174T cells. The negative controlswere negative. The capacity of the scFv and diabody produced in Pichiapastoris to identify human CEA on human tumor cells that express thisantigen fixed on polystyrene plates, by indirect immunofluorescence, wasdemonstrated.

Example 4 Purification of the ScFv and Diabody Produced in Bacteria

Procedure (a) Purification of the ScFv and Diabody Fragments, UsingImmobilized Ion Metal Affinity Chromatography (IMAC) and Ionic Exchange

The presence of the six histidines domain in the recombinant protein,donated by the vector pJG-1m, was used for purification. These sequencesconfer proteins a very high affinity for metallic ions (for exampleZn⁺², Cu⁺², Ni⁺²) that can be chelated to different chromatographicsupports, allowing an easy and reproducible purification.

The recombinant bacteria obtained as described before were centrifugedand the periplasm contents isolated through osmotic shock and briefsonication (seconds), and after dialyzed for 72 hours in coupling buffer(Tris-HCl 20 mM, 1 M NaCl, 20 mM Imidazole, pH 7.0). The bacterialperiplasm preparations containing the scFv and the diabody were applieddirectly and independently to a Sepharose-IDA-Cu⁺² matrix (Pharmacia).Once the proteins were coupled, the gels were first washed with 10 timestheir volume using coupling buffer, followed in a similar fashion withwash buffer (Tris-HCl 20 mM, 1 M NaCl, 150 mM Imidazole, pH 7.0) toeliminate E. coli contaminant proteins. The elution of the scFv and thediabody was done with Tris-HCl 20 mM, 1 M NaCl, 250 mM Imidazole, pH7.0. The samples of the elution peaks were submitted to a 12% SDS-PAGEto verify the presence of the proteins of interest. The eluted fractionscontaining the scFv and the diabody were concentrated in UltraFree 15(Amicon) devices, were dialyzed in a buffer solution containing Tris-HCl20 mM, pH 8.7, and were submitted to a second step of purification usingionic exchange. For this, the samples were applied to a Mono Q column(Pharmacia), and eluted through a linear NaCl gradient (0 to 1 M). Thesamples of the collected peaks were checked in 12% SDS-PAGE. Thepresence of the scFv and diabody at the expected sizes (approximately 27kDa) was verified. The final achieved purity for the two molecules wasvery similar and close to 95%, estimated through SDS-PAGE and silverstaining. The peaks of pure scFv and diabody were concentrated inUltraFree 15 (Amicon) devices up to 2 mg/mL. The biological activity ofthe purified preparations was verified using ELISA, following aprocedure similar to the one described previously in this invention. Allthe samples were conserved at 4° C.

Procedure (b) Analysis of the ScFv and Diabody through Gel Filtration

The scFv and diabody purified as described by the previous procedurewere studied using molecular sieve chromatography to determine thehomogeneity of the samples and the presence of multimers. Superdex 200(Pharmacia) was used for this, and a conventional process of gelfiltration in a HPLC equipment. It was determined that the scFvconcentrated in a major peak of approximately 27 kDa, correspondent to amonomeric form. The diabody appeared mainly with a size of approximately45 kDa, corresponding to a dimeric form.

Example 5 Characterization of the Diabody through Proteolytic Digestionand Mass Spectrometry

The purified diabody was dialyzed overnight at 4° C. against a buffersolution containing 1% NH₄HCO₃ at pH=8.3, containing Urea at aconcentration of 2 mol/L. The dialyzed protein was digested withsequence grade trypsin (Promega) in an enzyme:substrate ratio of 1:50during 4 hours at 37° C. The proteolytic digestion was arrested byacidification with equal volume of an aqueous solution of 1% trifluoroacetic acid, and stored at −20° C. until the moment of analysis byliquid chromatography coupled to the mass spectrometer (LC-MS).

The tryptic digestions were separated by reverse-phase chromatography ina liquid chromatograph AKTA Basic (Amersham Pharmacia Biotech) using alinear gradient from 0% to 80% of solution B in 100 minutes. Thesolutions used to generate the gradient were: A: H₂O/TFA 0.05% and B:Acetonitrile/TFA 0.05%.

The fractions obtained during the proteolytic digestion were analyzed bymass spectrometry using electrospray ionization (ESI-MS), by the way ofconnecting on line with the chromatographic system a LC-MS hybrid massspectrometer with orthogonal geometry QTOF-2 (Micromass Ltd.). Duringthe LC-MS measurement, the mass spectra were acquired from 350 to 1800in 0.98 seconds and using 0.02 seconds between each scanning. The massspectrometer was calibrated with a saline solution composed by a mixtureof sodium and cesium iodide. The voltages used in the cone and thecapillary were of 50 and 3000 volts, respectively. The spectra wereprocessed using the programs package MassLinx v 3.5 (Micromass Ltd).

In FIG. 4 and its adjunct Table the chromatographic profile of thetryptic digestion of the diabody, and the summary of the assignation ofthe tryptic peptides of the diabody, can be seen. In the ESI-MS spectrano signals indicating incorrectly linked cysteines were detected,evident from the summaries of the fractions 8 and 12 of the Tableadjunct to FIG. 4, that contains the peptides(²⁰Phe-Arg³¹)-S—S-(⁸⁷Ser-Arg⁹⁷) and (¹⁴³Val-Lys¹⁴⁸)-S—S-(¹⁸⁶Ile-Lys²²⁸)linked by disulphide bonds (—S—S—) between cysteines 22 and 95, and 147and 212, respectively.

From the peptides analyzed by ESI-MS, 92% of the diabody sequence couldbe obtained in a single proteolytic digestion (FIG. 5). In this sequencethere is a full coincidence with the aminoacid sequences deducted fromthe base sequence of the VH and VL domains (SEQ ID No. 16 and 17),amplified by PCR starting from the total RNA of the hybridoma thatproduces the Mab CB/ior-CEA.1, of the 5-aminoacid linker segment (SEQ IDNo. 10), and in the C-terminal portion, of the sequence of the c-mycpeptide and 6 final histidines, provided by vector pJG-1m (FIG. 2).

Example 6 Studies of Recognition of Deglycosylated CEA

Human CEA (Calbochem) was enzymatically deglycosylated with theendoglycosidase PNGasa F (New England Biolabs) specific forN-glycosylation. The CEA was dissolved in phosphate buffer 20 mM pH 7.8and denatured with SDS and 2-mercaptoethanol at 100° C. for 5 min. NP-40and 1 μL of PNGasa F were then added for 2 hours at 37° C. The controland deglycosylated samples were analyzed in SDS-PAGE with Coomasie bluestaining resulting in a significant reduction of molecular size (closeto 50%) after digestion with the endoglycosidase. A Western blot wascarried out using (a) Mab CB/ior-CEA.1, (b) the purified divalent scFv(diabody) or (c) an anti-human CEA antiserum obtained in mouse, asprimary antibodies, followed by polyclonal antibodies against the Fab ofMab CB/ior-CEA.1 conjugated to horseradish peroxidase for (a) and (b),and anti-mouse IgG antibodies conjugated to horseradish peroxidase for(c). The transference and development were similar as describedpreviously in this invention for Western blots. The Mab CB/ior-CEA.1 andthe diabody only recognized the non deglycosylated antigen. Thepolyclonal antiserum recognized CEA before and after deglycosylation.

Samples of native human CEA were analyzed through a Dot Blot system forrecognition of specific lectins. The employed lectins were the Sambucusnigra agglutinin (SNA) and the Maackia amurensis agglutinin (MAA),specific for terminal syalic acid, linked alpha 2,6 and alpha 2,3,respectively. The lectins employed in these experiments had beenconjugated with Digoxigenin, which is identified by an anti-Digoxigeninantibody labeled with alkaline phosphatase. The samples positive for theinteraction of lectin-oligosaccharide were developed by reaction with asubstrates specific for phosphatase (a mixture of 4-nitro bluetetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate). Fetuinwas used in this experiment as positive control from both lectins. Thenative CEA was recognized by SNA and not by MAA, a fact that indicated ahigh prevalence of terminal syalic acids linked alpha 2,6.

The human CEA was then digested with the enzyme NANAsa II, andexoglycosidase (syalidase) able to hydrolyze the terminal syalic acidsalpha 2,6. The products of digestion were separated in SDS-PAGE, and thestudy of their recognition was done by Western blot, using as primaryantibodies the Mab CB/ior-CEA.1 and an anti-human CEA antiserum obtainedin mouse. The results indicated that the native CEA control wasrecognized by both samples, while only the mouse anti-CEA antiserumrecognized the CEA digested with NANAsa II.

Example 7 Immunocyto- and Histochemical Study in Human Normal and TumorTissues

The tissues study was done in samples selected from normal and tumortissue archives, coming from autopsy material. A minimum panel oftissues was used to verify the recognition already described for MabCB/ior-CEA.1 (Tormo B et al. APMIS 97: 1073-1080, 1989). The specimensincluded: carcinomas of the lung, skin, breast, cervix, esophagus andkidney, adenocarcinomas of colon, prostate, pancreas, gall bladder,small intestine and stomach, tumors of neural, hematopoietic andsarcomatous origin, as well as normal colon mucosa, and normal tissuesas liver, kidney, lung, testicle, spleen and pancreas, including alsoblood cells.

The study was done following procedures previously reported (Tormo B etal. APMIS 97: 1073-1080, 1989), with some variations. The tissuespecimens were fixed in 10% buffered formalin, dehydrated, cleared andembedded in paraffin according to routine procedures. The histopathologywas evaluated in sections colored with hematoxilin-eosin. Consecutivesections of the blocks evaluated by histopathology were used for theimmunoperoxidase technique.

Paraffin-free, re-hydrated sections were treated with 3% H₂O₂ for 30minutes to block endogenous peroxidase, washed in phosphate bufferedsaline (PBS), and incubated with the samples, these diluted in PBS-1%bovine serum albumin (dilution buffer), for one hour. Then, the slideswere incubated for 30 minutes with a 1:100 dilution of biotinylatedpolyclonal IgG rabbit antibodies, obtained by immunization with the Fabof Mab CB/ior-CEA.1, and finally for a similar time with a 1:500dilution of a peroxidase-streptavidin complex (Amersham).

The examined simples were:

-   -   (a) Mab CB/ior-CEA.1 (positive control) at a concentration of 20        μg/mL    -   (b) E. coli purified scFv, as described in EXAMPLE 4, procedure        (a), at a concentration of 50 μg/mL    -   (c) E. coli purified diabody, as described in EXAMPLE 4,        procedure (a), at a concentration of 50 μg/mL    -   (d) The previously obtained scFv, denominated “F3” in these        Examples (Ayala et al. Biotechniques 13: 790-799, 1992; Pérez L        et al. Applied Biochem. Biotechnol. 24: 7982, 1996), purified        and at concentrations of 50 and 100 μg/mL

All dilutions were done in dilution buffer, and the incubations at roomtemperature, in humid chamber. Between steps, 3 washings of 1 minuteeach with dilution buffer or PBS were done. The immunoperoxidasereaction was developed via a 5-10 minute incubation with a solution thatcontained 3 mg of diaminobencidine, 5 mL of PBS and 5 mL of 30% H₂O₂.The slides were counter-stained with Meyer's hematoxilin. Thecharacteristic brown color reaction was registered as: negative orpositive, in three increasing intensity levels (1+, 2+, 3+). In eachslide the labeling was done with the sample in question, and in anadjoining zone with the dilution buffer as negative control.

For the studies in blood cells, the erythrocytes were first removed, andremnant white cells were applied on glass slides coated with gelatin,and fixed with acetone:methanol 1:1 (v:v). The rest of the technique wasdeveloped basically as described above.

The obtained results are summarized in Table VI, with respect to thestudied tissue. The normal tissues studied (liver, kidney, lung,testicles, blood, spleen, pancreas) were not identified by the fragmentsor by the Mab. In the case of the colonic mucosa, and in coincidencewith what had been obtained before for Mab CB/ior-CEA.1, the F3 scFv,and the new scFv and diabody, reacted exclusively with the luminalsecretion products and in the apical zones of some glands. The intensityof the reaction in the case of the F3 scFv was lower, something that wasalso later seen for several tumors. In the case of the blood cells, theMab, the scFv and diabody did not show reaction with normal lymphocytesand neutrophils, indicating the absence of an important cross reactivitywith the NCA antigen. On the contrary, the F3 scFv showed some minorrecognition of these cells.

The Mab, the scFv, and the diabody reacted with most of the tumors ofgastrointestinal origin, and the strong labeling was observed in themajority of the cases both in the apical surface of tumor cells, and inthe cytoplasm. None of these samples labeled tumors of hematopoietic andsarcomatous origin, or others derived from epithelium, exception made ofa canalicular breast carcinoma, and a lung large cell carcinoma. In thewell differentiated colon adenocarcinomas the labeling was intense inthe apical zone of the cytoplasm, and in the luminal secretion products,while in the moderately and poorly differentiated adenocarcinomas thelabeling was observed in all cytoplasm. Exception made of very fewsamples, the staining intensities were very similar for these threemolecules.

In the case of F3, a general lowering of the staining intensity wasseen, even though in some occasions, concentrations two times higherthan those used for the scFv and diabody were employed. The lowerintensity of staining could have caused that some samples identified bythe other antibodies were not recognized by F3. TABLE VI Immunocyto- andhistochemical study with different antibodies. Tissues scFv F3 scFvDiabody CB/ior-CEA.1 Normal: lung 0/2 0/2 0/2 0/2 thymus 0/2 0/2 0/2 0/2kidney 0/2 0/2 0/2 0/2 liver 0/2 0/2 0/2 0/2 spleen 0/2 0/2 0/2 0/2testicle 0/2 0/2 0/2 0/2 colon 2/2 (a)* 2/2* 2/2* 2/2* blood 0/2 (b) 0/20/2 0/2 Tumor: ADC stomach 1/2 (a) 2/2 2/2 2/2 ADC pancreas 1/3 (a) 2/32/3 2/3 ADC gall bladder 0/1 1/1 1/1 1/1 Ca esophagus 0/1 0/1 0/1 0/1ADC intestine 1/2 (a) 2/2 (a) 2/2 2/2 Ca lung (large cells) 1/1 (a) 1/11/1 1/1 ADC colon 5/6 (a) 6/6 (c) 6/6 6/6 (BD, MD, PD) Ca breast 0/1 1/11/1 (a) 1/1 ADC prostate 0/2 0/2 0/2 0/2 Ca cervix 0/2 0/2 0/2 0/2 Cakidney 0/1 0/1 0/1 0/1 Ca skin 0/1 0/1 0/1 0/1 HDG Lymphoma 0/1 0/1 0/10/1 non-HDG Lymphoma 0/1 0/1 0/1 0/1 Rhabdomyosarcoma 0/1 0/1 0/1 0/1Liposarcoma 0/1 0/1 0/1 0/1Note:The numbers in the Table represent the cases with positivestaining/total number of cases studied. If no brackets are present, theintensity of staining in the positives was between 2+ and 3+;Ca: carcinoma;ADC: adenocarcinoma;HDG: Hodgkin;BD: well differentiated;MD: medium differentiated;PD: poorly differentiated.*the labeling appears circumscribed to the products of luminal secretionand apical zones of some glands;(a) the positive cases had staining that could be classified as 1+;(b) recognition of lymphocytes and eosinophils with intensity that couldbe classified as 1+;(c) 2 of the 6 positive cases showed staining with intensity that couldbe classified as 1+.

Example 8 Determination of Affinity Constant

For the determination of the affinity constant an ELISA no competitivemethod (Beatty J D et al. J. Immunol Meth. 100: 173-184, 1987) based onthe mass action law, was used. The affinity constant Kaff is equal to[AgAb]/[Ag][Ab], where AgAb is the antigen-antibody complex in L/mol(M⁻¹), [Ag] is the concentration of free antigen (mol), and [Ab] is theconcentration of free antibody (mol).

Four double serial dilutions of human CEA (Calbochem) were used in thecoating of polyvinyl ELISA plates (Costar). The plates were blockedusing PBS-skim milk 1%. The samples (scFv F3, scFv, diabody, MabCB/ior-CEA.1, all purified) were applied to the plates at variousconcentrations. After washings, the wells corresponding to the threefirst samples were incubated with the Mab 9E10 (10 μg/mL), while inthose corresponding to Mab CB/ior-CEA.1 blocking solution was used. Inthe following step an anti mouse IgG antibody conjugated to peroxidase(Sigma) was added in dilution 1:2500 for one hour, at 37° C. The usedsubstrate was OPD, and the reaction was developed for 15 minutes. Thereading of absorbance was done at 492 nm in a LabSystems Multiskan MSequipment.

The optical density (OD) values for each case were plotted in theordinate axis (y), and the concentration in ng/mL in the abscissa axis(x), in a logarithm base 10 scale. OD 100 was taken as that at which thesignal was maintained at a maximum. For each curve, half of the OD 100(OD 50) was calculated. The concentration values of each sample at OD 50were determined for each curve, and the affinity calculations carriedout with the following formula: Kaff=(n−1)/2(n), where n=[Ab′]t/[Ab]t.[Ab′]t is the concentration value of the sample that corresponds to anOD 50 value for the highest antigen concentration to compare, and [Ab]tis the concentration value of the simple that corresponds to an OD 50value for the lowest antigen concentration to compare. The six possibleaffinity determinations for the 4 obtained curves, estimating the finalKaff as the average of these.

Table VII reflects the Kaff values calculated for each the assayedvariants. The scFv has a Kaff of (5.0±0.4)×10⁹ L mol⁻¹, a magnitude morethat one order and a half higher than that obtained for F3(Kaff=(9.2±0.8)×10⁷ L mol⁻¹). This last value basically corresponds withthe calculated for F3 in measurements made by a different procedure(Pérez L et al. Applied Biochem. Biotechnol. 24: 79-82, 1996). Thediabody has a Kaff of (2.8±0.3)×10¹⁰L mol⁻¹, while that for the MabCB/ior-CEA.1 the Kaff value was (6.1±0.5)×10¹⁰L mol⁻¹. TABLE VII Valuesof affinity calculated for the developed experiments. Assayed sampleKaff (L mol⁻¹) Mab CB/ior-CEA.1 (6.1 ± 0.5) × 10¹⁰ Diabody (2.8 ± 0.3) ×10¹⁰ scFv (5.0 ± 0.4) × 10⁹ ScFv F3 (9.2 ± 0.8) × 10⁷Kaff: affinity constant, calculated as the average of six determinations± standard deviation (in brackets).

Example 9 Determination of Specific In Vivo Recognition of Fragments andAntibody Labeled with ¹²⁵I, in C57BI/6 Mice Bearing Tumors Induced bythe Inoculation of B16-CEA13 Cells

For the determination of the in vivo specific recognition of theantibody fragments, the following molecules were labeled with ¹²⁵I(Amersham, UK) using the Iodogen method (Fraker P J, Speck J C Jr.Biochem Biophys Res Comm 80:849-857, 1978):

-   -   (a) scFv purified from E. coli; (specific activity 1.1 MBq/5 μg)    -   (b) diabody purified from E. coli; (specific activity: 1.2 MBq/5        μg)    -   (c) Mab CB/ior-CEA.1; (specific activity: 1.8 MBq/5 μg)    -   (d) Purified ScFv F3 (Ayala et al. Biotechniques 13: 790-799,        1992; Pérez L et al. Applied Biochem. Biotechnol. 24:        79-82, 1996) (specific activity: 1.0 MBq/5 μg).

The radiolabeled products were analyzed in thin layer chromatography todetermine the incorporation to protein, and values between 95 and 98% ofthe radioactivity were found. The capacity of the radiolabeled productsto detect CEA was assayed in a system where polystyrene tubes werecoated with CEA (5 μg/mL; Calbochem), blocked, and samples of theradiolabeled products added, adjusted to the amounts of antibody thatcould be entrapped by this solid phase. After incubation a washing, itwas determined that 80, 79, 83, and 81% of the radioactivity wasentrapped by the solid phase, respectively, for the samples (a)-(d)described above, demonstrating that the radiolabeling procedure did notsensibly affect the biological activity of the antibodies.

To study the biodistribution, 4 groups of animals were formed, each of12 C57BI/6 mice (CENPALAB, Cuba). The animals were inoculated with 1×10⁶B16-CEA13 cells per animal, using the intra-axillary route. The tumorswere visible and palpable (approximately 0.3-0.5 g) after 7 days, afterwhich the mice were injected with the radiolabeled product in questionby the tail vein, and sacrificed after 12, 24 and 48 hours, withsurgical removal of the tumor and the following normal tissues: spleen,liver, kidney, intestine, muscle, bone marrow, and blood. Theaccumulation of radioactivity was expressed as percentage of theinjected dose per gram of tissue. The calibration was done through astandard sample of the injected dose. Radioactivity was determined usinga gamma scintillation counter.

The B16-CEA13 cells used in these experiments were obtained through thetransfection of a gene that encodes for the extracellular domains ofhuman CEA, cloned in the pDisplay™ vector (Cat. No. V660-20,Invitrogen). The gene was obtained by PCR from RNA extracted fromCRL-1682 cells, with oligonucleotides designed alter the publishedsequence of human CEA. The recombinant plasmid pDisplay-CEA was purifiedand transfected into C57BI/6 mouse B16-F10 melanoma cells (ATCCCRL-6475) using Lipofectamine PLUS™ (Gibco-BRL) and 5 μg of DNA pertransfection. The selection of stable transfectants was done with 4.0mg/mL of geneticyn sulphate (G418; Gibco-BRL) for 14 days, after whichthe surviving cultured cells were cloned by limiting dilution and thoseclones that expressed human CEA in their surface were identified throughindirect immunofluorescence, using Mab CB/ior-CEA.1 as first antibody,and anti mouse IgG antibodies conjugated with FITC (Sigma) fordevelopment. It was found that 73% of the clones presented more than 80%of the cells with specific membrane fluorescence indicating that thehuman CEA was exposed correctly folded and glycosylated in theirsurface.

B16-F10 non transfected cells were employed as controls. The replicas ofthe clones selected as positive through indirect immunofluorescence weremultiplied and injected independently into C57BI/6 mice, 1×10⁶ cells peranimal, using the intra-axillary route. Of the 10 clones that gave riseto tumors, the one with faster and progressive growth characteristics,denominated B16-CEA13, was selected for the experiments reported here.

FIG. 6 shows the percentage of radioactivity recovered per studiedtissue, at different times (with respect to the injected total), and theratio radioactivity in the tumor:radioactivity in blood. The resultsincluded in Table VIII demonstrate that between 24 and 48 hours, theratio radioactivity in the tumor:radioactivity in blood maintains highfor the diabody, the scFv, and the Mab, with the highest values for thelatter, followed by the dimeric molecule. The F3 scFv obtainedpreviously showed very low values, with an in vivo inadequate behaviorthat can be correlated with its reduced affinity for CEA. TABLE VIIIRatio radioactivity in tumor:radioactivity in blood for C57BI/6 micetransplanted with the mouse B16-CEA13 melanoma, that expresses humanCEA. The values correspond to 24 and 48 hours alter the animals wereinjected with the different molecules radiolabeled with ¹²⁵I. Each ratiowas calculated from the mean values derived from the tissues recoveredfrom 12 mice. Molecule 24 hours 48 hours scFv 43.60 53.50 diabody 47.1061.17 ScFv F3 1.79 2.62 Mab CB/ior-CEA.1 51.70 71.30

1. An antibody fragment of the monomeric scFv type obtained from the RNAextracted from the hybridoma producing Mab CB/ior-CEA.1, that isspecific for human carcinoembryonic antigen (CEA) either in solubleform, adsorbed to solid surfaces, or present in cells, and shows anaffinity constant for CEA of (5.0±0.4)×10⁹ L mol⁻¹ and a recognition forsuch antigen dependent on the conservation of its glycosylation.
 2. Anantibody fragment of the monomeric scFv type according to claim 1,comprising an aminoacid sequence set forth in SEQ ID No
 16. 3. Anantibody fragment of the divalent (diabody) scFv type obtained from theRNA extracted from the hybridoma producing Mab CB/ior-CEA.1, that isspecific for human carcinoembryonic antigen (CEA) either in solubleform, adsorbed to solid surfaces, or present in cells, and shows anaffinity constant for CEA of (2.8±0.3)×10¹⁰ L mol⁻¹ and a recognitionfor such antigen dependent on the conservation of its glycosylation. 4.An antibody fragment of the divalent (diabody) scFv type according toclaim 3, comprising an aminoacid sequence set forth in SEQ ID No
 17. 5.Antibody fragments according to claim 1 for the identification of tumorcells that express human CEA.
 6. Recombinant or synthetic recombinantantibodies specific for human CEA comprising the aminoacidic sequencesof the variable domains VH and VL in SEQ ID 16 and SEQ ID 17, linkedartificially in the form of Fab fragments and other scFv variants,bispecific antibodies, or fused to biologically or biochemically activedomains.
 7. Antibody fragments according to claim 1 wherein thefragments are produced in recombinant bacteria or yeast, in insect ormammalian transfected cells, or in genetically modified organisms. 8.Antibody fragments according to claim 1 further comprising a radioactivelabel or detectable by other method, or a chemical or biological agentwith antitumor potential.
 9. Pharmaceutical composition comprisingantibody fragments according to claim 1, for the treatment of humantumors that express CEA.
 10. Pharmaceutical composition comprisingantibody fragments according to claim 1, for the in vivoradiolocalization of human tumors that express CEA, using imagingtechniques.
 11. Reagent for the in vitro or ex vivo diagnosis comprisingantibody fragments according to claim 1, for the detection of human CEA,linked or not to cells.
 12. Cells that express antibody fragmentsaccording to claim 1, obtained through genetic manipulation by way ofrecombinant DNA, being these cells bacteria, yeast, insect cells,mammalian cells, or plant cells.
 13. Multicellular organisms thatexpress antibody fragments according to claim 1, obtained throughgenetic manipulation by way of recombinant DNA, being these organismstransgenic animal or transgenic plants.
 14. Vectors that encode forantibody fragments according to claim 1, obtained through geneticmanipulation by way of recombinant DNA, being these vectors plasmids orsequences able to integrate in host cells.
 15. Antibody fragmentsaccording to claim 3 for the identification of tumor cells that expresshuman CEA.
 16. Antibody fragments according to claim 3 wherein thefragments are produced in recombinant bacteria or yeast, in insect ormammalian transfected cells, or in genetically modified organisms. 17.Antibody fragments according to claim 6 wherein the fragments areproduced in recombinant bacteria or yeast, in insect or mammaliantransfected cells, or in genetically modified organisms.
 18. Antibodyfragments according to claim 3 further comprising a radioactive label ordetectable by other method, or a chemical or biological agent withantitumor potential.
 19. Antibody fragments according to claim 6 furthercomprising a radioactive label or detectable by other method, or achemical or biological agent with antitumor potential. 20.Pharmaceutical composition comprising antibody fragments according toclaim 3, for the treatment of human tumors that express CEA. 21.Pharmaceutical composition comprising antibody fragments according toclaim 6, for the treatment of human tumors that express CEA. 22.Pharmaceutical composition comprising antibody fragments according toclaim 3, for the in vivo radiolocalization of human tumors that expressCEA, using imaging techniques.
 23. Pharmaceutical composition comprisingantibody fragments according to claim 6, for the in vivoradiolocalization of human tumors that express CEA, using imagingtechniques.
 24. Reagent for the in vitro or ex vivo diagnosis comprisingantibody fragments according to claim 3, for the detection of human CEA,linked or not to cells.
 25. Reagent for the in vitro or ex vivodiagnosis comprising antibody fragments according to claim 6, for thedetection of human CEA, linked or not to cells.
 26. Cells that expressantibody fragments according to claim 3, obtained through geneticmanipulation by way of recombinant DNA, being these cells bacteria,yeast, insect cells, mammalian cells, or plant cells.
 27. Cells thatexpress antibody fragments according to claim 6, obtained throughgenetic manipulation by way of recombinant DNA, being these cellsbacteria, yeast, insect cells, mammalian cells, or plant cells. 28.Multicellular organisms that express antibody fragments according toclaim 3, obtained through genetic manipulation by way of recombinantDNA, being these organisms transgenic animal or transgenic plants. 29.Multicellular organisms that express antibody fragments according toclaim 6, obtained through genetic manipulation by way of recombinantDNA, being these organisms transgenic animal or transgenic plants. 30.Vectors that encode for antibody fragments according to claim 3,obtained through genetic manipulation by way of recombinant DNA, beingthese vectors plasmids or sequences able to integrate in host cells. 31.Vectors that encode for antibody fragments according to claim 6,obtained through genetic manipulation by way of recombinant DNA, beingthese vectors plasmids or sequences able to integrate in host cells.